The chemical energy for sperm motility is provided by the hydrolysis of ATP within the flagellum. To elucidate the mechanism of sperm motility, it is necessary to isolate, characterize, and localize the movement-related ATPase of the flagellum. To extract this ATPase(s) from mammallian sperm, uncontaminated by ATPase from membranes and disrupted mitochondria, procedures were developed for the preparation of plasmalemmae-free, mitochondria-free flagella isolated from bull sperm. Transmission electron microscopy of thin sectioned samples, used to evaluate the effect of each treatment on internal flagellar ultrastrucure, showed alterations in some movement-related structures within the flagellum. Therefore, we have developed a radiomicroassay which will enable stoichiometric accounting of ATPase activity throughout the sperm dissection. This accounting of ATPase activity will enable better correlation of biochemical and ultrastructural alterations and will be useful in monitoring the isolation and purification of the flagellar ATPases.